Method for treating meibomian gland disease

ABSTRACT

A method for treating a patient having meibomian gland disease, ocular irritation associated with delayed tear clearance, or recurrent corneal epithelial erosion, is disclosed. Preferably, the method concerns treatment of a patient with topical tetracycline, a derivative or analogue of tetracycline, or a chemically modified tetracycline (CMT). Oral administration of a CMT is also disclosed as part of the method for treating meibomian gland disease, ocular irritation associated with delayed tear clearance, or recurrent corneal epithelial erosion.

BACKGROUND OF THE INVENTION

Meibomian gland disease is the most common tear film and ocular surfacedisorder causing eye irritation. The incidence of the disease increaseswith age, and occurs in approximately 50% of patients with the skindisease, rosacea. A conservative estimate of the number of patientsaffected with this condition is 10 million in the United States alone.It has been reported that 15% of patients with ocular rosacea developrecurrent corneal epithelial erosions, a potentially sight-threateningproblem.

Common complaints of patients suffering from meibomian gland diseaseinclude blurred or filmy vision, burning or foreign body sensations inthe eye, photophobia, and pain severe enough to awaken the person fromsleep. Although patients with this condition usually have normalproduction of aqueous tears by their lacrimal glands, their meibomianglands can atrophy and this is frequently accompanied by metaplasia ofthe ductal epithelium of these glands. Anterior erosion of themucocutaneous junction of the eyelid is often noted, as well as eyelidand conjunctival infection, eyelid margin irregularity, cornealepithelial changes, and corneal vascularization.

The mechanisms responsible for the eyelid and ocular surface changes andirritation symptoms experienced by patients with meibomian gland diseasewere heretofore unknown. Therefore, previous treatments of meibomiangland disease were directed only to treatment of presumed infection ofthe eyelids or meibomian glands, or had particular disadvantages thatmade such treatments of little use for long periods of time. Forexample, patients with meibomian gland disease have been symptomaticallytreated with artificial tears, but these agents provide limited, if any,improvement. Topically applied steroids to the eyelids or ocular surfaceare effective as short-term pulse therapies. However, steroids are notgood long-term solutions because of the potential side-effects e.g.,cataract and glaucoma. Meibomian gland disease is currently not curableor reversible; therefore, patients with this condition must be treatedfor life.

Orally administered tetracyclines and tetracycline analogues (e.g.,doxycycline and minocycline) having antibiotic activity are commonly andeffectively used for prophylactic or therapeutic treatment of meibomiangland disease. The mechanism by which tetracyclines work in treatingmeibomian gland disease is not known, but some relief of symptoms hasbeen reported. However, one disadvantage for using antimicrobiallyactive tetracyclines or tetracycline analogues orally in the treatmentof meibomian gland disease is that a high percentage of patients areunable to tolerate oral tetracyclines for extended periods of time. Theintolerance to tetracyclines can manifest itself in gastrointestinalproblems, e.g., epigastric pain, nausea, vomiting, and diarrhea, orother problems related to taking long-term oral antibiotics, such asmucosal candidiasis. At the present time there are no availablelong-term treatments of meibomian gland disease.

Recently, tetracycline and tetracycline analogues have been reported tohave antimicrobial-inflammatory effects (e.g., reduction of IL-1 andnitric oxide production) and to inhibit synthesis and activation ofMMPs. Tetracyclines have been reported to be effective in treatingdiseases where chronic inflammation and tissue destruction due toincreased collagenase activity have been implicated. These diseasesinclude rheumatoid arthritis, sterile cornealulceration, andperiodontitis.

Certain modifications of the chemical structure of tetracycline resultin a tetracycline analogue which lacks antimicrobial activity. Thesenon-antimicrobial, chemically modified tetracyclines (CMTs) retain theiranti-inflammatory and anti-collagenolytic activities. Topicaladministration of antimicrobial tetracyclines or non-antimicrobial CMTs,or oral administration of non-antimicrobial CMTs represent a majoradvance in the therapy of patients having meibomian gland diseasebecause both treatment modalities eliminate systemic toxicity and wouldallow lifetime administration of, these therapeutic agents for thischronic disease.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a class of agents and methods of usingthose agents for treatment of a patient having meibomian gland disease,including relief of symptoms or conditions associated with the disease,such as ocular irritation, delayed tear clearance, or recurrent cornealepithelial erosion. More specifically, the subject invention concernsagents and methods for decreasing ocular irritation, and surfaceinflammation, improving tear clearance, reducing tear IL-1αconcentration, or inhibiting IL-1α-mediated matrix metalloproteinaseactivity which is increased in patients with meibomian gland disease ordelayed tear clearance. The subject invention can thus be useful forreducing eye irritation, improving tear clearance, reducing IL-1αconcentration in the tear fluid, or inhibiting MMP activity in the tearfluid of patients with delayed tear clearance and increased tear fluidIL-1α.

In a preferred embodiment, the subject invention concerns use of aneffective amount of a topically administered antimicrobial tetracyclineor tetracycline analogue, or a non-antimicrobial analogue oftetracycline. Non-antimicrobial tetracycline analogues are commonlyreferred to and accepted in the scientific literature as “chemicallymodified tetracyclines” (CMTs). These compounds, or compositionscomprising those compounds, can be used for treating meibomian glanddisease, ocular irritation associated with delayed tear clearance, orrecurrent corneal epithelial erosion. Alternatively, CMTs can beadministered orally for treating meibomian gland disease, ocularirritation associated with delayed tear clearance, or recurrent cornealepithelial erosion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IL-1α concentrations in tear fluid for rosacea patients,ideal controls, and age-matched control subject groups.

FIG. 2 shows gelatinase activity plotted against IL-α concentrations fornormal control (NL) patients and meibomian gland disease (MGD) patients.

FIG. 3 shows fluorescein tear concentration plotted against IL-αconcentration for normal control (NL) patients and meibomian glanddisease (MGD) patients.

FIG. 4 shows fluorescein tear concentration plotted against gelatinaseactivity normal control (NL) patients and meibomian gland disease (MGD)patients.

FIG. 5 shows the concentration (pg/ml) of mature IL-1α in supernatantsof human corneal epithelial cell cultures. Media=negative control;LPS=cultures treated with lipopolysaccharide for 24 h; LPS MP=culturestreated with LPS and the corticosteroid methylprednisolone for 24 h; LPSDoxy=cultures treated with LPS and doxycycline (5 μg/ml) for 24 h.

FIG. 6 shows the ratio of the mature to precursor forms of IL-1α insupernatants of human corneal epithelial cell cultures. Media=negativecontrol; LPS=cultures treated with lipopolysaccharide for 24 h; LPSMP=cultures treated with LPS and the corticosteroid methylprednisolonefor 24 h; LPS Doxy=cultures treated with LPS and doxycycline (5 μg/ml)for24 h.

FIG. 7 shows the concentration (μg/ml) of IL-1RA in supernatants ofhuman corneal epithelial cell cultures. Media=negative control;LPS=cultures treated with lipopolysaccharide for 24 h; Doxy=culturestreated with doxycycline (5 μg/ml) for 24 h; CMT 3=cultures treated withchemically modified tetracycline CMT 3 (1 μg/ml) for 24 hr;Doxy+LPS=cultures treated with LPS and doxycycline (5 μg/ml) for 24 h;CMT 3+LPS=cultures treated with LPS and chemically modified tetracyclineCMT 3 (1 μg/ml) for 24 hr.

FIG. 8 is a gelatin zymogram of human corneal epithelial cellsupernatant: doxycycline treatment and MMP-3 activation. Lane Legend:(1) 92 kDa purified pro-MMP-9 standard (0.1 ng); (2) Corneal epithelialsupernatant+50 μg/mL doxycycline; (3) Supernatant+50 μg/mLdoxycycline+1.5 mg/μL MMP-3; (4) Supernatant+100 μg/mL doxycycline+1.5ng/μL MMP-3; (5) Supernatant from cell culture pre-treated withdoxycycline; (6) Supernatant from cell culture pre-treated withdoxycycline+1.5 ng/μL MMP-3.

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns, in a preferred embodiment, the use of topicallyapplied antimicrobial tetracyclines, including tetracycline or atetracycline analogue, or topically or orally administeredchemically-modified tetracyclines (CMTs) for treatment of a patienthaving meibomian gland disease. According to the subject invention,treatment of a patient having meibomian gland disease:includes reducingor reversing irritation symptoms, delayed tear clearance, recurrentcorneal epithelial erosion or aqueous tear deficiency. The CMTs lackantimicrobial activity. For purposes of this invention, tetracycline andits analogues having antimicrobial activity are termed “antimicrobialtetracyclines” and the tetracycline analogues lacking antimicrobialactivity are termed “CMTs”.

We recently discovered that patients with meibomian gland disease havesignificantly delayed tear clearance, rapid tear break-up time (ofteninstantaneous), and tear film instability. In addition, it was foundthat meibomian gland disease patients have significantly greaterconcentrations of the pro-inflammatory cytokine, interleukin I alpha(IL-1α) in their tear fluid.

IL-1α has been reported to have multiple biological effects, includingincreasing expression of adhesion molecules by vascular endothelial andepithelial cells that facilitate binding of inflammatory cells, inducingexpression of chemotactic cytokines such as interleukin 8, stimulatingproduction of prostaglandins that increase vascular permeability andpain, stimulating production of nitric oxide that causes vasodilationand redness, and stimulation of production of matrix-degrading enzymes,termed matrix metalloproteinases (MMPs), by several different celltypes, including mucosal epithelium.

We found that the activity of one member of the MMP family, MMP-9 (alsotermed gelatinase B), is markedly increased in the tear fluid ofpatients with meibomian gland disease associated with rosacea, ascompared to normal asymptomatic patients. In addition, activity ofgelatinase B appears to be inversely correlated with tear clearance.

These data indicate that antimicrobial tetracyclines and tetracyclineanalogues and CMTs possess biological activities capable of neutralizingthe inflammatory and matrix degrading factors that have been found to bestrongly correlated with the development of ocular rosacea in ourstudies.

Advantageously, topically applied tetracyclines or CMTs can have greaterefficacy because of the higher concentrations that can be achieved atthe disease site. Because of their lack of antimicrobial-bacterialeffect and greater therapeutic activity, CMTs can have fewer systemic orother side effects than tetracyclines, whether administered orally ortopically.

Topical administration of a tetracycline analogue in an ointment (e.g.,oxytetracycline) or in solution (e.g., doxycycline eye drops), topatients having meibomian gland disease associated with rosacea resultedin complete resolution of symptoms in 73% (8 of 11) of the patientstreated with the tetracycline analogue ointment and more than 90% (10 of11) of the patients treated with the tetracycline analogue solution.

Several patients who had previously used oral tetracycline stated thatimprovement in their symptoms was better with topically appliedoxytetracycline than orally administered tetracycline. Three patientshad moderate but not complete relief of symptoms. Three other patientsexperienced burning from the medication, one of which ceased treatment.

The tetracycline utilized in the present invention may be any of thereadily available, pharmaceutically acceptable tetracyclines known inthe medical art. Included in this group of tetracyclines are those suchas chlortetracycline, which is marketed under the tradename Acronize,Aureocina, Aureomycin, Biomitsin, Biomycin, and Chrysomykine;Demeclyeycline marketed as Lederrnycin, Detravis, Meciclin, andMexocine; Doxycyline marketed as Vibramycin, Vibramycin Hyclace,Liomycin Vibradox, Panamycin, Titradox, Hydramycin, Tecacin; Lymecyclinewhich is marketed as Armyl, Mucomycin, Tetramyl, Tetralysal;Methacycline which is marketed as Adriamicina, Cyclobiotic,Germicilclin, Globociclina, Megamycine, Pindex, Londomycin, Optimycin,Rondomycin; Minocycline which is marketed as Minocin, Klinomycin,Vectrin; Oxytetracycline which is marketed as Biostat, Oxacycline,Oxatets, Oxydon, Oxymycin, Oxytan, Oxytetracid, Ryomycin, Stezazin,Tetraject, Terramycin, Tetramel, Tetran, Dendarcin, Dendarcin;Rolitetracycline marketed as Bristacin, Reverin, Superciclin; Syntetrex,Syntetrin, Synotodecin, Tetraverin, Transcycline, Velacicline,Velacycline; and Tetracycline marketed as Achromycin, Ambramycin,Cyclomycin, Polycycline, Tetrabon, and Tetracyn.

The active salts which are formed through protonation of thedimethylamino group on carbon atom 4, exist as crystalline compounds andare very stable in water. However, these amphoteric antibiotics willcrystallize out of aqueous solutions of their salts unless stabilized byan excess of acid. The hydrochloride salts are used most commonly fororal administration. Water soluble salts may be obtained also from basessuch as sodium or potassium hydroxides but are not stable in aqueoussolution, they are also formed with divalent and polyvalent metals.

The tetracyclines or CMTs used according to the present invention arepreferably orally administered at a dosage level from about 10% to about100%, and preferably about 20% to about 80% of the normal antibiotictherapeutic dose of the particular tetracycline compound being employed.By normal antibiotic therapeutic dose is meant the dosage of theparticular tetracycline compound which is commonly used and recommendedfor the treatment of bacterial infection.

Alternatively, sub-antimicrobial dose means a dose having no significantantimicrobial effect in vitro or in vivo. More than 100% of the normalantibiotic therapeutic dose can be utilized in the method of the presentinvention. The normal antibiotic therapeutic dose of the tetracyclineis, for the most part, well studied and well documented.

Tetracyclines and chemically modified tetracyclines for topicaladministration can be added to buffered and electrolyte balanced aqueoussolutions, buffered and electrolyte balanced aqueous solutions with alubricating polymer, mineral oil or petrolatum-based ointment, otheroils, liposomes, cylcodextrins, sustained release polymers or gels in arange from 0.001 to 3% (weight per volume or weight per weight). Thesepreparations can be administered to the preocular tear film or onto theeyelid skin or lid margin 1 to 6 times per day for a period up to thelifetime of the patient. For example, an eye drop solution comprisingdoxycycline as an active ingredient was prepared by dissolvingpharmaceutical grade doxycycline hydrochloride powder in anelectrolyte-balanced salt solution (BSS

, Alcon, Ft. Worth, Tex.) to a final concentration of 0.025%.

In a preferred embodiment, the compounds useful according to the subjectinvention are tetracyclines which have been chemically modified so as tosubstantially reduce or eliminate antimicrobial properties and increasetheir antimicrobial-inflammatory activity. Methods for reducingantimicrobial properties of a tetracycline are disclosed in TheChemistry of the Tetracyclines, Ch. 6, Mitscher, Ed., at p. 211. Aspointed out by Mitscher, modification at positions 1, 2, 3, 4, 10, and12a can lead to loss of antimicrobial activity. Such chemically modifiedtetracyclines (CMTs) are included in the preferred embodiment of thepresent invention, since they can be used without disturbing the normalmicrobial flora of the treated mammal as would happen with extendedexposure to antimicrobial tetracyclines.

Preferable CMTs used according to the subject invention include thoselacking a dirnethylamino side chain at position 4. For example,4-dedimethylamino-tetracycline, 4-dedimethylamino-5-oxytetracycline,4-dedimethylamino-7-chlortetracycline,4-hydroxy-4-dedimethylaminotetracycline,4-dedimethylamino-12a-deoxytetracycline,4-dedimethylamino-11-hydroxy-12a-deoxytetracycline,4-dedimethylamino-7-dimethylaminotetracycline,6-dimethyl-6-deoxy-4-dedimethylaminotetracycline,6-o-deoxy-5-hydroxy-4-dedimethyla minotetracycline,11a-chlortetracycline, 12a-deoxytetracycline, and the 2-nitrilo analogsof tetracycline.

The amount of tetracycline or CMT administered to effectively treatmeibomian gland disease, ocular irritation associated with delayed tearclearance, or recurrent corneal epithelial erosion is an amount thatsignificantly improves tear clearance, reduces IL-1α concentration inthe tear fluids, or IL-1α mediated production of MMPs, or MMP activity.The maximal dosage for humans is the highest dosage that does not causeclinically important side effects. For the purpose of the presentinvention, side effects include clinically important disruption of thenormal flora as well as toxic effects to the eye surface.

The dosage of tetracyclines administered in the present invention isalso additionally dependent upon the age and weight of the person beingtreated, the mode of administration, and the type and severity of theinflammatory or matrix-degrading disease being treated. For illustrativepurposes, a suitable amount of the antimicrobial tetracycline,doxycycline, is 0.1-4.0 mg/kg/day. In the case of a non-antimicrobialtetracycline, for example, the dose for 4-dedimethylaminotetracyclinecan be 0.1-30 mg/kg/day. However, in either case, the preferred methodof treatment includes tetracycline or CMT compositions administeredtopically to the eye in suitable pharmaceutical carriers. Thepharmaceutical carrier may be in the form of a solution, suspension,ointment, gel, or other form suitable for topical administration of thetetracycline or topical or oral administration of CMT to the affectedmammal. For oral administration, the CMTs utilized according to thesubject invention can be formulated in the form of tablets, capsules,elixirs, or the like. Additionally, the tetracyclines or CMTs of thepresent invention can be reasonably incorporated into a polymer carrierdelivery system for use topically, subconjunctivally, or orally. Inaddition, other means of administration are contemplated, such as byinjection, either intramuscularly or intravenously.

The following Examples describe in detail compositions and methodsillustrative of the present invention. It will be apparent to thoseskilled in. the art that many modifications, both of materials andmethods, may be practiced without departing from the purpose and intentof this disclosure.

EXAMPLE 1 Identification of Increased IL-1α Concentrations in MeibomianGland Disease Patients

In one study, tear fluid concentrations of Interleukin-1-alpha (IL-1α),Tumor Necrosis Factor-α (TNF-α), and Epidermal Growth Factor (EGF) inpatients having ocular rosacea were compared with those concentrationsin normal patients (controls).

Fourteen (14) patients with severe meibomian gland disease, facialrosacea, and symptoms of ocular irritation were examined for ocularsurface disease, tear production and tear clearance rate (TCR). Formeibomian gland disease assessment, meibomian glands were examined byslit lamp biomicroscopy and graded for the presence of orificemetaplasia, expressibility of meibum, and meibomian gland acinardropout, as previously described and known from the scientificliterature. Twelve (12) controls, frequency-matched for age, and fifteen(15) ideal normals were assessed using the same parameters.

Minimally stimulated tear samples (20 μl) were drawn from each subjectand analyzed using a sandwich ELISA to detect IL-1α, TNF-α, and EGF.Sandwich ELISA assays for EGF, IL-1α and TNF-α were performed usingcommercial kits (R&D Systems, Minneapolis, Minn.). Prior to eachanalysis tears were-extracted in a masked fashion by the laboratorytechnician from the saturated rods by centrifuging at 3,500 g for 5minutes within the pipette tip.

The samples from both eyes of each patient were pooled. The rods andpipette tips were carefully removed and the tears aspirated (2 μl forEGF assay and 4 μl for IL-1α and TNF-α assays) and diluted in ELISAbuffer (supplied by manufacturer) to a final volume of 200 μl. The tearsamples were transferred to wells of microliter plate and the ELISAperformed according to the instructions provided by the manufacturer.Cytokine concentrations were determined from the standard curve asdescribed previously in the literature. The results of the cytokineconcentration assays for each subject group, are shown in Table 1 below.

TABLE 1 Inflammatory Cytokines in Tears Tumor Necrosis Interleukin-1αSubject Group Factor-α (pg/ml) (pg/ml) Rosacea patients <10 pg/ml 45.4 ±4.6 Age-matched controls <10 pg/ml 22.6 ± 5.0 (p = 0.003) Ideal controls<10 pg/ml 17.1 ± 3.4 (p < 0.001) P values listed for each control groupare derived from comparison of the mean value of that group with themean value of the same variable in the rosacea group.

Tear IL-1α concentration was significantly higher in rosacea patientsthan in age-matched (p=0.003) and ideal normal controls (p<0.001) (FIG.1). TNF-α was not detected in patients or controls, indicating levels ofless than 10 pg/ml. EGF was not significantly higher in rosacea patientsthan in age-matched controls. Tear Clearance LN(TCR) was significantlylower in rosacea patients than in both age-matched (p=0.048) and idealcontrols (p=0.002). Schirmer I scores were statistically lower inrosacea patients than ideal controls (p=0.013), but not age-matchedcontrols. IL-1α was correlated inversely with tear clearance LN(TCR)(r=0.58 p<0.0001) and Schirmer I (r=−0.39, p=0.012).

It was concluded that concentrations of IL-1α are present in normaltears but are elevated in ocular rosacea, whereas TNF-α is not Thereduced tear clearance LN(TCR), its inverse correlation with IL-lα, andthe absence of TNF-α the tears of these patients, suggests that theincreased concentration of IL-lα observed, may be largely due to failureof clearance of cytokine normally produced at the ocular surface.

In summary, there is a differential increase in the level of theinflammatory cytokine IL-1α in the tear fluid in patients having ocularrosacea. Much of this elevation can be caused by reduced tear clearance,which can encourage tear stagnation and perpetuate ocular surfaceinflammation by stimulating production of inflammatory molecules.

EXAMPLE 2 Correlation of Gelatinase Activity with IL-1α Concentrationand Tear Clearance

Tear fluorescein clearance was correlated with. IL-1α concentration and92 kD gelatinase (MMP 9) activity in the tears of patients. Thirteenpatients with ocular rosacea (including 1 patient with recurrentepithelial erosion, 2 with recurrent peripheral corneal infiltrates andvascularization and 2 patients with epithelial basement membranedystrophy) and 13 normal subjects with normal aqueous tear productionand no irritation symptoms were evaluated. Tear fluorescein clearancewas evaluated by measuring fluorescence in tear fluid collected from theinferior meniscus 15 minutes after instillation of 5 ml of 2%Na-fluorescein with a Cytofluor II. IL-1α was measured by ELISA using anR&D Systems kit. Gelatinase activity was evaluated by gelatinzymography, comparing tear activity to purified 92 kD gelatinase (MMP9).

Compared to normal controls, patients with ocular rosacea had greaterdelay of fluorescein clearance (p<0.001), higher tear IL-1αconcentration (p<0.001). and greater 92 kD gelatinase activity(p<0.001). Tear IL-1α concentration was correlated with gelatinaseactivity (rho=0.58, p<0.002) (FIG. 2); delayed tear clearance wascorrelated with elevated tear IL-1α concentration (rho=0.84, p<0.001)(FIG. 3) and increased gelatinase activity (rho=0.84, p<0.001) (FIG. 4).

EXAMPLE 3 Reduced Tear Clearance in Ocular Irritation

Reduced tear clearance is commonly found in most patients with ocularirritation irrespective of the patient's tear production. Forty (40)abnormal patients presenting with a chief complaint of ocular irritationand forty (40) asymptomatic controls of similar age distribution wereused to correlate and compare a new method of measuring tear fluoresceinclearance and the Schirmer 1 test with the severity of ocular irritationsymptoms, presence of meibomian gland disease, corneal fluoresceinstaining scores, and corneal and conjunctival sensitivity. All subjectscompleted a symptom questionnaire, a baseline ocular examination,fluorescein clearance test (FCT) and Schirmer test.

Methods. The fluorescein clearance test (FCT) was performed by measuringthe fluorescein concentration in minimally-stimulated tear samplescollected from 1.5 the inferior tear meniscus 15 minutes afterinstillation of 5 μl of 2% sodium fluorescein with a Cytofluor IIfluorophotometer. Severity of ocular irritation was assessed with asymptom questionnaire.

Schirmer 1 test, biomicroscopic meibomian gland evaluation, cornealfluorescein staining score and corneal and conjunctival sensation scoreswith the Cachet-Bonnet anesthesiometer were assessed in all subjects.

Results. Irritation symptoms correlated with higher log tear fluoresceinconcentration (symptomatic 3.08+/−0.62 units/μl, normal control1.89+/−0.7 units/μl, p<0.005) and lower Schirmer 1 test scores(symptomatic 12.6 mm, normal control 22.3 mm, p<0.005). The fluoresceinclearance test showed greater predictive value for identifying ocularirritation than the Schirmer 1 test.

EXAMPLE 4 Doxycycline Therapy Improves Tear Clearance and Reduces IL-1Concentration in the Tear Fluid

Six patients were treated with oral doxycycline 50 mg orally twice a dayfor 3 weeks. An improvement in irritation symptoms was observed in 5 of6 patients. Additionally, decreased conjunctival hyperemia and cornealfluorescein staining was observed in all patients. An improvement influorescein clearance was noted in 5 of 6 patients and reduction of tearfluid IL-1 concentration was also observed in 5 of 6 patients. Thesefindings indicate that tetracyclines reduce two of the significant riskfactors for ocular irritation in patients with meibomian gland disease.

Example 5 Resolution of Symptoms of Meibomian Gland Disease by TopicalLS Administration of Oxytetracycline

Eleven (11) patients with ocular irritation symptoms dub to meibomiangland disease associated with rosacea and delayed tear clearance weretreated with a commercially available oxytetracycline ointment (Terak,Akorn Pharmaceuticals) as a topical administration to the ocular surfaceor the eyelids. Seventy-three percent (73%) of these patients hadcomplete resolution of irritation symptoms. See Table 2 below.

TABLE 2 Patients with Ocular Rosacea Treated with OxytetracyclineOintment Patient's Clinical Number Initials BPEI # Date Treated ResponseComments 1 L P 410606 Jan. 2, 1997 E 2 H B 393796 Jan. 13, 1997 E 3 H P214814 Dec. 23, 1996 E 4 B P 358105 Dec. 2, 1996 G mild burning 5 J S547453 Jan. 10, 1997 G burned and stopped 6 F C 225688 Jan. 7, 1997 E 7J I 422804 Jan. 13, 1997 E 8 R R 401297 Jan. 13, 1997 E 9 C A 88261 Jan.27, 1997 E 10 D L 531404 Jan. 21, 1997 G mild burning 11 E S 554804 Dec.16, 1996 E E = Excellent response, complete or almost complete relief ofsymptoms. G = Good response, symptoms moderately, but not completedrelieved

Several patients who had previously used oral tetracycline stated thatimprovement in their symptoms was better with topically appliedoxytetracycline than orally administered tetracycline. Three patientshad moderate but not complete relief of symptoms. Three other patientsexperienced burning from the medication, one of which ceased treatment.

EXAMPLE 6 Resolution of Ocular Irritation by Administration of TopicalDoxycycline Solution

Moderate to complete relief of ocular irritation symptoms was alsoobserved in 10 of 11 patients experiencing ocular irritation due tomeibomian gland disease and delayed tear clearance that were treatedwith topical administration of a 0.025% aqueous solution of doxycyclineto their eyes 3 to 4 times per day for 2 weeks. See Table 3. Thesolution was prepared as described herein. A reduction of cornealfluorescein staining was observed in several of these patients.

TABLE 3 Patients Treated With Topical Doxycycline No. BPEI # DIAGNOSISRESPONSE 1 585447 MGD, DTC complete 2 460606 MGD, DTC moderate, betterthan oral 3 537137 MGD, DTC compete, better than oral 4 606807 MGD, DTCcomplete 5 590395 MGD, DTC complete 6 593046 MGD, DTC moderate 7 503909MGD, DTC complete, better than oral 8 558105 MGD, DTC moderate, not asgood as oral 9 405797 MGD, DTC no effect 10 576264 MGD, DTC moderate 11562422 MGD, DTC complete MGD = Meibomian gland disease DTC = delayedtear clearance

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

EXAMPLE 7 Doxycycline Inhibits the Conversion of Precursor IL-1β to itsMature Form in Cultured Human Corneal Epithelium

The effect of Doxydycline was evaluated on the concentrations ofprecursor and mature IL-1β in the supernatants of human cornealepithelial cell cultures that were stimulated with lipopolysaccharide(LPS). Doxycycline significantly decreased the level of the maturebiologically active form of IL-1β compared to LPS-stimulated culturesthat were not stimulated with doxycycline (FIG. 5). Furthermore,doxycycline markedly decreased the ratio of mature to precursor IL-1β(FIG. 6).A decrease in the level of IL-1β mRNA in doxycycline-treatedcultures was also observed. These findings demonstrate that doxycyclineinhibits that synthesis and activation of the inflammatory cytokineIL-1β.

EXAMPLE 8 Doxycycline Increases Release of the Anti-Inflammatory Form ofIL-1, IL-1RA

e-1 receptor antagonist (IL-1RA) is a molecule produced by epithelialand inflammatory cells that binds to the type 1 IL-1 receptor but doesnot initiate signal transduction.

IL-1 RA has an anti-inflammatory effect by competing with thepro-inflammatory forms of IL-1 (IL-1α and IL-1β) for the type 1receptor. The addition of doxycycline or the chemically modifiedtetracycline CMT-3 to human corneal epithelial cell culturessignificantly increased the release of IL-1RA in to the media comparedto untreated cultures. Furthermore, the . concentration of IL-1RA incorneal epithelial cultures stimulated with LPS and treated with eitherdoxycycline or CMT-3 was greater than cultures exposed to LPS alone(FIG. 7). These findings indicate tetracyclines are anti-inflammatorydue to their ability to stimulate IL-1RA release by the human cornealepithelium.

EXAMPLE 9 Doxycycline Decreases MMP-9 Activity in Human CornealEpithelial Cultures

Exposure of human corneal epithelial cultures to doxycycline (5• g/ml)for 24 hours decreased the level of pro-MMP9 activity in the supernatantby 70% compared to untreated cultures (FIG. 8). MMP-9 is themetalloproteinase responsible for initiating sterile corneal ulcerationand it is also capable of cleaving precursor IL-1β into its mature form.

All cited references are hereby incorporated herein by reference.

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1. A method of treating a patient having meibomian gland disease,aqueous tear deficiency, delayed tear clearance or recurrent cornealepithelial erosion, said method comprising: topically administering aneffective amount of tetracycline to an eye of the patient.
 2. The methodof claim 1, wherein said tetracycline is an antimicrobial tetracycline.3. The method of claim 2, wherein said antimicrobial tetracycline isoxytetracycline.
 4. The method of claim 2, wherein said antimicrobialtetracycline is doxycycline.
 5. The method of claim 1, wherein saidtetracycline is a non-antimicrobial tetracycline.
 6. The method of claim5, wherein said non-antimicrobial is a member selected from the groupconsisting of 4-dedimethylaminotetracycline,4-dedimethylamino-5-oxytetracycline,4-dedimethylamino-7-chlorotetracycline,4-hydroxy-4-dedimethylaminotetraycline,4-dedimethylamino-12a-deoxytetracycline,4-dedimethylamino-11-hydroxy-12a-deoxytetracycline,4-dedimethylamino-7-dimethylaminotetracycline,6-dimethyl-6-deoxy-4-dedimethylaminotetracycline,6-o-deoxy-5-hydroxy-4-dedimethylaminotetracycline,11a-chlortetracycline, 12a-deoxytetracycline and 2-nitrilo analogs oftetracycline.
 7. The method of claim 1, wherein said treatment increasestear clearance in the eye of the patient.
 8. The method of claim 1,wherein said treatment comprises inhibiting a member selected from thegroup consisting of matrix metalloproteinase activity in tear fluid,synthesis and activation of interleukin -1β, conversion of precursorinterleukin-1β to mature interleukin-1β, ocular surface inflammation andreactive oxygen species in tear fluid and ocular surface epithelium. 9.The method of claim 8, wherein said matrix metalloproteinase comprisesmatrix metalloproteinase-9.
 10. The method of claim 1, wherein saidtreatment comprises increasing production of interleukin-1 receptorantagonist by corneal epithelium.
 11. The method of claim 1, whereinsaid treatment comprises reducing interleukin-1-α concentration in tearfluid.
 12. A method of treating a patient having meibomian glanddisease, aqueous tear deficiency, delayed tear clearance or recurrentcorneal epithelial erosion, said method comprising: orally administeringan effective amount of tetracycline to an eye of the patient.
 13. Themethod of claim 12, wherein said tetracycline is an antimicrobialtetracycline.
 14. The method of claim 13, wherein said antimicrobialtetracycline is oxytetracycline.
 15. The method of claim 13, whereinsaid antimicrobial tetracycline is doxycycline.
 16. The method of claim12, wherein said tetracycline is a non-antimicrobial tetracycline. 17.The method of claim 16, wherein said non-antimicrobial is a memberselected from the group consisting of 4-dedimethylaminotetracycline,4-dedimethylamino-5-oxytetracycline,4-dedimethylamino-7-chlorotetracycline,4-hydroxy-4-dedimethylaminotetracycline,4-dedimethylamino-12a-deoxytetracycline,4-dedimethylamino-11-hydroxy-12a-deoxytetracycline,4-dedimethylamino-7-dimethylaminotetracycline,6-dimethyl-6-deoxy-4-dedimethylaminotetracycline,6-o-deoxy-5-hydroxy-4-dedimethylaminotetracycline,11a-chlortetracycline, 12a-deoxytetracycline and 2-nitrilo analogs oftetracycline.
 18. The method of claim 12, wherein said treatmentincreases tear clearance in the eye of the patient.
 19. The method ofclaim 12, wherein said treatment comprises inhibiting a member selectedfrom the group consisting of matrix metalloproteinase activity in tearfluid, synthesis and activation of interleukin-1β, conversion ofprecursor interleukin-1β to mature interleukin-1β, ocular surfaceinflammation and reactive oxygen species in tear fluid and ocularsurface epithelium.
 20. The method of claim 19, wherein said matrixmetalloproteinase comprises matrix metalloproteinase-9.
 21. The methodof claim 12, wherein said treatment comprises increasing production ofan interleukin-1 receptor antagonist by corneal epithelium.
 22. Themethod of claim 12, wherein said treatment comprises reducinginterleukin-1-α concentration in tear fluid.
 23. A composition fortreating a patient having meibomian gland disease, said compositioncomprising an antimicrobial tetracycline or a non-antimicrobialtetracycline as an active ingredient in a balanced salt solution. 24.The composition of claim 23, wherein said active ingredient has a finalconcentration of between about 0.001% to about 1.0%.
 25. The compositionof claim 23, wherein said active ingredient has a final concentration ofabout 0.025%.
 26. The composition of claim 23, wherein said activeingredient is doxycycline.
 27. The composition of claim 23, wherein saidantimicrobial tetracycline comprises a member selected from the groupconsisting of a topical ointment, a gel and a sustained-releasepreparation.